Many examples of methods for assaying a target nucleic acid are methods for assaying a nucleic acid in a homogenous solution by using a fluorescent dye-labeled target nucleic acid probe having properties changing in fluorescence on hybridizing with a target nucleic acid (called a nucleic acid probe for a homogenous solution system or a target nucleic acid probe, having the same meaning). The method does not require, by this characteristics, 1) any step immobilizing a target nucleic acid (or any step washing a probe) indispensable for common hybridizing method and 2) any step washing out non-reacting probes (or any step immobilizing non-reacting genes); the method, therefore, is a method for assaying easily, rapidly and accurately a target nucleic acid. With such reasons, the method for assaying quantitatively a target nucleic acid has been in a wide used for various methods for analyzing genes (see non-patent reference 1).
In assaying directly a target nucleic acid by a nucleic acid probe for a homogenous solution system, an object could be achieved by using the following procedures.
(1) making a target nucleic acid of known concentrations ready beforehand hybridize with a nucleic acid probe for a homogenous solution system, and, on this hybridization, monitoring a change or the amount of change in an optical character;
(2) preparing a calculating curve for determining a target nucleic acid by preparing an equation relating to the above change or amount of change in an optical character and amounts of a target nucleic acid because the change or the amount of change is positively proportional to amounts of a target nucleic acid;
(3) conducting a procedures similar to the above procedures in regard to an unknown sample, and determining the amount of a target nucleic acid from the above calculating curve based on the obtained change or amount of change in an optical character.
In the method, however, if a target nucleic acid exists greater in concentration than an added nucleic acid probe, a change or the amount of a change in an optical character is at any time stationary. Because of this, the conventional methods need any of the means, 1) diluting a target nucleic acid sample, and 2) preparing in advance plural assaying systems with a probe having various concentrations. The above 1) requires a diluting processing step; the operation become complicated. As results, the above 2) has such problems that (1) long assaying time is needed; (2) diluting errors occur; and (3) on automation of the assaying, an for dilution is needed. In the above 2) also, the reaction time and reaction temperature suitable for hybridization vary with response to the concentrations of an added probe (if a target nucleic acid and a target nucleic acid probe are higher in concentrations, the time for completed hybridization becomes shorter; if contrary to the former, the reaction time becomes longer. If a target nucleic acid and a probe are higher in concentrations, a Tm value is higher; if contrary to the former, it becomes lower); as a result, there is such problems that (1) an assaying system needs to be optimized in every concentration of a probe, and (2) a calculating curve needs to be prepared in every concentration of an added probe.
Non-patent reference 1: Protein, Nucleic Acid and Enzyme; vol. 35, No. 17, Kyoritu Shuppan Co., Ltd.; Experimental Medical; vol. 15, No. 7, 1997, Yodosha Co., Ltd.